Charged Hexosaminides as New Substrates for beta-N-Acetylhexosaminidase-Catalyzed Synthesis of Immunomodulatory Disaccharides

This work is a structure-activity relationship study that investigates the influence of the nature and amount of negative charge in carbohydrate substrates on the affinity of ?-N-acetylhexosaminidases, and on the stimulation of natural killer cells. It describes synthetic procedures yielding novel glycosides that are useful in immunoactivation. Specifically, we present a thorough study on the ability of six C-6 modified ?-N-acetylhexosaminides (aldehyde, uronate, 6-O-sulfate, 6-O-phosphate) to serve as substrates for cleavage and glycosylation by a library of ?-N-acetylhexosaminidases from various sources. Four novel disaccharides with one or two (negatively) charged groups were prepared in synthetic reactions in good yields. Surprisingly, the 6-O-phosphorylated substrate, although cleaved by a number of enzymes from the series, worked neither as a donor nor as an acceptor in transglycosylation reactions. The results of wet experiments were supported by molecular modeling of substrates in the active site of two representative enzymes from the screening. All ten prepared compounds were examined in terms of their immunoactivity, namely as ligands of two activation receptors of natural killer (NK) cells, NKR-P1 and CD69, both with isolated proteins and whole cells. Sulfated disaccharides in particular acted as very efficient protectants of NK cells against activation-induced apoptosis, and as stimulants of the natural killing of resistant tumor cells, which makes them good candidates for potential clinical use in cancer treatment.